Compositions and methods for modifying the content of polyunsaturated fatty acids in mammalian cells

ABSTRACT

The present invention features compositions (e.g, nucleic acids encoding fat-1, optionally and operably linked to a constitutively active or tissue-specific promoter or other regulatory sequence and pharmaceutically acceptable formulations including that nucleic acid or biologically active variants thereof) and methods that can be used to effectively modify the content of PUFAs in animal cells (i.e., cells other than those of  C. elegants , for example, mammalian cells such as myocytes, neurons (whether of the periferal or central nervous system), adipocytes, endothelial cells, and cancer cells). The modified cells, whether in vivo or ex vivo (e.g., in tissue culture), transgenic animals containing them, and food products obtained from those animals (e.g., meat or other edible parts of the animals (e.g., liver, kidney, or sweetbreads)) are also within the scope of the present invention.

[0001] This application claims priority from U.S. Ser. No. 60/275,222,filed Mar. 12, 2001, the contents of which are incorporated herein byreference in their entirety.

TECHNICAL FIELD

[0002] This invention relates to compositions and methods for alteringthe content of polyunsaturated fatty acids in mammalian cells.

BACKGROUND

[0003] Some of the work presented herein was supported by a grant fromthe National Institutes of Health (CA79553). The United Statesgovernment may, therefore, have certain rights in the invention.

[0004] Polyunsaturated fatty acids (PUFAs) are fatty acids having 18 ormore carbon atoms and two or more double bonds. They can be classifiedinto two groups, n-6 or n-3, depending on the position (n) of the doublebond nearest the methyl end of the fatty acid (Gill and Valivety, TrendsBiotechnol. 15:401-409, 1997; Broun et al., Annu. Rev. Nutr. 19:197-216,1999; Napier et al., Curr. Opin. Plant Biol. 2:123-127, 1999). The n-6and n-3 PUFAs are synthesized through an alternating series ofdesaturations and elongations beginning with either linoleic acid (LA,18:2n6) or α-linolenic acid (ALA, 18:3n3), respectively (Gill andValivety, supra; Broun et al., supra; Napier et al., supra). The majorend point of the n-6 pathway in mammals is arachidonic acid (AA, 20:4n6)and major end points of the n-3 pathway are eicosapentaenoic acid (EPA,20:5n3) and docosahexaenoic acid (DHA, 22:6n3).

[0005] An important class of enzymes involved in the synthesis of PUFAsis the class of fatty acid desaturases. These enzymes introduce doublebonds into the hydrocarbon chain at positions determined by the enzyme'sspecificity. Although, in most cases, animals contain the enzymaticactivity to convert LA (18:2n6) and ALA (18:3n3) to longer-chain PUFA(where the rate of conversion is limiting), they lack the 12- and15-desaturase activities necessary to synthesize the precursor (parent)PUFA, LA and ALA (Knutzon et al., J. Biol. Chem. 273:29360-29366, 1998).Furthermore, the n-3 and n-6 PUFA are not interconvertible in manmaliancells (Goodnight et al., Blood 58: 880-885, 1981). Thus, both LA and ALAand their elongation, desaturation products are considered essentialfatty acids in the human diet. The PUFA composition of mammalian cellmembranes is, to a great extent, dependent on dietary intake (Clandininet al., Can. J. Physiol. Pharmacol. 63:546-556, 1985; McLennan et al.,Am. Heart J. 116:709-717, 1988).

[0006] To the contrary, some plants and microorganisms are able tosynthesize n-3 fatty acids such as ALA (18:3n-3) because they havemembrane-bound 12- and 15-(n-3) desaturases that act on glycerolipidsubstrates in both the plastid and endoplasmic reticulum (Browse andSomerville, Annu. Rev. Plant Physiol. Plant Mol. Biol. 42: 467-506,1991). Genetic techniques have led to the identification of the genesencoding the 12- and 15-desaturases from Arabidopsis thaliana and otherhigher plant species (Okuley et al., Plant Cell 6:147-158, 1994; Arondelet al., Science 258:1353-1355, 1992). Recently, a fat-1 gene encoding ann-3 fatty acid desaturase was cloned from Caenorhabditis elegans(Spychalla et al., Proc. Natl. Acad. Sci. USA 94:1142-1147, 1997; seealso U.S. Pat. No. 6,194,167).

SUMMARY

[0007] The present invention is based, in part, on the discovery thatthe C. elegans n-3 desaturase gene, fat-1, can be successfullyintroduced into other types of animal cells (e.g., mammalian cells),where it quickly and effectively elevates the cellular n-3 PUFA contentand dramatically balances the ratio of n-6:n-3 PUFAs. More specifically,heterologous expression of the fat-1 gene in rat cardiac myocytesrendered those cells capable of converting various n-6 PUFAs to thecorresponding n-3 PUFA and changed the n-6:n-3 ratio from about 15:1 (anundesirable ratio) to 1:1 (a desirable ratio). In addition, aneicosanoid derived from n-6 PUFA (i.e. arachidonic acid) wassignificantly reduced in the trasgenic cells (as described furtherbelow, levels of arachidonic acid can be assessed to determine whether agiven nucleic acid encodes a biologically active desaturase; similarly,one can assess the levels of n-6 PUFA; the levels of n-3 PUFA; and theratio of n-6:n-3 PUFAs). Accordingly, the present invention featurescompositions (e.g., nucleic acids encoding fat-1, optionally andoperably linked to a constitutively active or tissue-specific promoter)and methods that can be used to effectively modify the content of PUFAsin animal cells (i.e., cells other than those of C. elegans, forexample, mammalian cells such as myocytes, neurons (whether of theperipheral or central nervous system), adipocytes, endothelial cells,and cancer cells). More generally, a fat-1 sequence or a biologicallyactive variant thereof can be operably linked to a regulatory sequence.Regulatory sequences encompass not only promoters, but also enhancers orother expression control sequence, such as a polyadenylation signal,that facilitates expression of the nucleic acid. The modified cells(whether in vivo or ex vivo (e.g., in tissue culture)), transgenicanimals containing them, and food products obtained from those animals(e.g., meat or other edible parts of the animals (e.g., liver, kidney,or sweetbreads)) are also within the scope of the present invention.

[0008] In one embodiment, the invention features mammalian cells thatcontain a nucleic acid sequence encoding the C. elegans n-3 desaturaseor biologically active variants (e.g., fragments or other mutants)thereof. Biologically active variants of the n-3 desaturase enzyme arevariants that retain enough of the biological activity of a wild-typen-3 desaturase to be therapeutically or clinically effective (i.e.,variants that are useful in treating patients, producing transgenicanimals, or conducting diagnostic or other laboratory tests). Forexample, variants of n-3 desaturase can be mutants or fragments of thatenzyme that retain at least 25% of the biological activity of wild-typen-3 desaturase. For example, a fragment of an n-3 desaturase enzyme is abiologically active variant of the full-length enzyme when the fragmentconverts n-6 fatty acids to n-3 fatty acids at least 25% as efficiencyas the wild-type enzyme does so under the same conditions (e.g., 30, 40,50, 75, 80, 90, 95, or 99% as efficient as wild-type n-3 desaturase).Variants may also contain one or more amino acid substitutions (e.g.,1%, 5%, 10%, 20%, 25% or more of the amino acid residues in thewild-type enzyme sequence can be replaced with another amino acidresidue). These substitutions can constitute conservative amino acidsubstitutions, which are well known in the art. Cells that express afat-1sequence (optionally, operably linked to a constitutively active ortissue-specific promoter) are valuable aids to research because theyprovide a convenient system for characterizing the functional propertiesof the fat-1 gene and its product (cells in tissue culture areparticularly convenient, but the invention is not so limited). They alsoallow one to study any cellular mechanism mediated by n-3 fatty acidswithout the lengthy feeding procedures of cells or animals that arecurrently required, and they serve as model systems that can be used,for example, to evaluate existing methods and to design new methods foreffectively transferring sequences encoding an n-3 desaturase into cellsin vivo. In any of these contexts (e.g. whether the compositions of theinvention are being used to treat patients, to generate transgenicanimals, or in cell culture assays), nucleic acids encoding fat-1 or abiologically active variant thereof can be co-expressed (by way of thesame or a separate vector) with a heterologous gene. The heterologousgene can be, for example, another therapeutic gene (e.g., a receptor fora small molecule or chemotherapeutic agent) or a marker gene (e.g., asequence encoding a fluorescent protein, such as green fluorescentprotein (GFP) or enhanced (EGFP)).

[0009] The nucleic acids of the invention can be formulated foradministration to a patient. For example, they can be suspended insterile water or a physiological buffer (e.g., phosphate-bufferedsaline) for oral or parenteral administration to a patient (e.g.,intravenous, intramuscular, intradermal, or subcutaneous injection (inthe event the patient has a tumor, the compositions can be injected intothe tumor or adminstered to the tissue surrounding the site from which atumor was removed) or by inhalation).

[0010] The invention also features transgenic animals (including anyanimal kept as livestock or as a food source) that express the C.elegans n-3 desaturase gene or a biologically active variant thereof.Given the discovery that a C. elegans fat-1 gene can be efficientlyexpressed when delivered to a mammalian cell, this gene can be used togenerate transgenic mice or larger transgenic animals (such as cows,pigs, sheep, goats, rabbits or any other livestock or domesticatedanimal) according to methods well known in the art. Depending on whetherthe construct used contains a constitutively active promoter or atissue-specific promoter (e.g., a promoter that is active in skeletalmuscle, breast tissue, the colon, neurons, retinal cells, pancreaticcells (e.g., islet cells) etc.) the fat-1 gene can be expressed globallyor in a tissue-specific manner. The cells of the transgenic anrimalswill contain an altered PUFA content that, as described further below,is more desirable for consumption. Thus, transgenic livestock (or anyanimal that is sacrificed for food) that express the desaturase enzymeencoded by the fat-1 gene will be superior (i.e., healthier) sources offood. Food obtained from these animals can be provided to healthyindividuals or to those suffering from one or more of the conditionsdescribed below.

[0011] As noted, the invention features methods of treating patients(including humans and other mammals) who have a condition associatedwith an insufficiency of n-3 PUFA or an imbalance in the ratio ofn-3:n-6 PUFAs by administering a nucleic acid encoding an n-3 desaturaseor a biologically active variant thereof (e.g., a fragment or othermutant). Alternatively, one can administer the protein encoded. Themethods can be carried out with patients who have an arrhythmia orcardiovascular disease (as evidenced, for example, by high plasmatriglyceride levels or hypertension), cancer (e.g., breast cancer orcolon cancer), inflammatory or autoimmune diseases (such as rheumatoidarthritis, multiple sclerosis, inflammatory bowel disease (IBD), asthma,chronic obstructive pulmonary disease, lupus, diabetes, Sjogren'ssyndrome transplantation, ankylosing spondylitis, polyarteritis nodosa,reiter's syndrome, and scleroderma), a malformation (or threatenedmalformation, as occurs in premature infants) of the retina and brain,diabetes, obesity, skin disorders, renal disease, ulcerative colitis,Crohn's disease, chronic obstructive pulmonary disease, or who are atrisk of rejecting a transplanted organ. Given that fat-1 expression canalso inhibit cell death (by apoptosis) in neurons, the methods of theinvention can also be used to treat or prevent (e.g., inhibit thelikelihood of, or the severity of) neurodegenerative diseases.Accordingly, the invention features methods of treating a patient whohas (or who may develop) a neurodegenerative disease such as Parkinson'sdisease, Alzheimer's disease, Huntington's disease (HD), spinal andbulbar muscular atrophy (SBMA; also known as Kennedy's disease),dentatorubral-pallidoluysian atrophy, spinocerebellar ataxia type 1(SCA1), SCA2, SCA6, SCA7, or Machado-Joseph disease (MJD/SCA3) (Reddy etal. Trends Neurosc. 22:248-255, 1999). As a balanced n-6:n-3 ratio isessential for normal growth and development, and as noted above, themethods of the invention can be advantageously applied to patients whohave no discernable disease or condition.

[0012] Abbreviations used herein include the following: AA forarachidonic acid (20:4n-6); DHA for docosahexaenoic acid (22:6n-3); EPAfor eicosapentaenoic acid (20:5n-3); GFP for green fluorescent protein;Ad.GFP for adenovirus carrying GFP gene; Ad.GFP.fat-1 for adenoviruscarrying both fat-1 gene and GFP gene; and PUFAs for polyunsaturatedfatty acids.

[0013] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, useful methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflicting subject matter, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

[0014] Other features and advantages of the invention will be apparentfrom the following detailed description, the drawings, and the Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a collection of four photomicrographs showing genetransfer efficiency. Rat cardiac myocytes were infected with Ad.GFP(left panels; control) or Ad.GFP.fat-1 (right panels). Forty-eight hoursafter infection, cardiomyocytes were visualized with bright light (upperpanels) and at 510 nm of blue light (lower panels). Coexpression of GFPdemonstrates visually that the transgene is being expressed in cellswith a high efficiency.

[0016]FIG. 2 is an autoradiogram of a ribonuclease (RNase) protectionassay of fat-1 transcript levels in cardiac myocytes infected withAd.GFP (control) and myocytes infected with Ad.GFP.fat-1. Total RNA (10μg) isolated from the cardiomyocytes was hybridized with anti-sense RNAprobes, digested with RNase and resolved by electrophoresis through adenaturing polyacrylamide gel. The fat-1 mRNA was visualized byautoradiography. A probe targeting β-actin gene was used as control.

[0017]FIG. 3. is a pair of partial gas chromatograph traces showingfatty acid profiles of total cellular lipids extracted from controlcardiomyocytes infected with Ad.GFP and cardiomyocytes infected withAd.GFP.fat-1.

[0018]FIG. 4 is a bar graph depicting prostaglandin E₂ levels in controlcardiomyocytes and cardiomyocytes expressing the fat-1 gene (asdetermined by enzyme immunoassay). Values are means ±SDs of threeexperiments and are expressed as % of control. *p<0.01.

[0019]FIG. 5 is a Table showing the polyunsaturated fatty acidcomposition of total cellular lipids from control cardiomyocytes and thetransgenic cardiomyocytes expressing a C. elegans fat-1 cDNA.

[0020]FIG. 6 is a flowchart of an experimental protocol.

[0021]FIG. 7 is a flowchart of an experimental protocol.

[0022]FIG. 8 is a flowchart of an experimental protocol.

[0023]FIG. 9 is a pair of partial gas chromatograph traces showing fattyacid profiles of total cellular lipids extracted from control neuronsand neurons infected with Ad-GFP-fat-1. FIG. 10 is a Table comparing thePUFA composition of total cellular lipids from rat cortical neurons(control) and transgenic cells expressing a C. elegans fat-1 cDNA(fat-1).

[0024]FIG. 11 is a bar graph showing the results of an enzymeimmunoassay of prostaglandin E₂ levels in control neurons and neuronsexpressing the fat-1 gene. Ad-GFP-fat-1 infected neurons have lowerlevels of PGE₂ relative to control. Values are means ±SD of threeexperiments and expressed as a percentage of control. *P<0.01.

[0025]FIG. 12 is a bar graph representing the results of an MTT assay ofcell viability in control and fat-1 expressing cultures. After 24 hoursof growth factor withdrawal, the cell viability of neurons expressingthe fat-1 gene is 50% higher than control cells (p<0.01).

[0026]FIG. 13 is a pair of tracings showing differential responses ofmyocytes infected with Ad.GFP and myocytes infected with Ad.GFP.fat-1 to7.5 mM extracellular calcium.

[0027]FIG. 14 is a line graph showing tumor volume over time (0-4 weeksafter viral injection) and thus, the effect of gene transfer on tumorgrowth. Breast cancer cells (MDA-MB-231) were implanted subcutaneouslyon the back of nude mice. Three weeks later, the mice were treated withAd.GFP-fat-1 or Ad.GFP (control; 50 μl, 10¹² VP/m) by intratumoralinjection.

[0028]FIG. 15 is a table showing PUFA compositions of total cellularlipids from control MCF-7 cells and the transgenic MCF-7 cellsexpressing a C. elegans fat-1 cDNA.

[0029]FIG. 16 is a bar graph depicting the results of an enzymeimmunoassay of prostaglandin E₂ levels in control MCF-7 cells and MCF-7cells expressing fat-1 gene. Values are means ±SE of three experimentsand expressed as a percentage of control. (*P<0.05).

[0030]FIGS. 17A and 17B are representations of the nucleotide sequenceof the C. elegans fat-1 cDNA (FIG. 18A) and the deduced amino acidsequence of the Fat-1 polypeptide (FIG. 18B).

DETAILED DESCRIPTION

[0031] The studies described below demonstrate that, inter alia, anucleic acid molecule encoding an n-3 desaturase can be efficientlyexpressed in a variety of mammalian cell types and, as a consequence,those cells produce significant amounts of n-3 PUFA from endogenous n-6PUFA and have a more balanced ratio of n-6 to n-3 PUFA (1:1). Thestudies were carried out using recombinant adenoviral expressionvectors, which can mediate gene transfer in vivo or in vitro. Adenoviralvectors expressing fat-1, or biologically active variants thereof, aswell as other types of viral and non-viral expression vectors are withinthe scope of the invention now claimed. Other viral vectors that can beemployed as expression constructs in the present invention includevectors derived from viruses such as vaccinia virus (e.g., a pox virusor a modified vaccinia virus ankara (MVA)), an adeno-associated virus(AAV), or a herpes viruses. These viruses offer several attractivefeatures for various mammalian cells. For example, herpes simplexviruses (e.g., HSV-1) can be selected to deliver fat-1 or a biologicallyactive variant thereof, to neuronal cells (and thereby treat patientswith neurodegenerative conditions).

[0032] Other retroviruses, liposomes, and plasmid vectors are also wellknown in the art and can also be used (e.g., the expression vectorpUR278 can be used when one wishes to fuse a fat-1 sequence to the lacZgene; lacZ encodes the detectable marker β-galactosidase (see, e.g.,Ruther et al., EMBO J., 2:1791, 1983). A fat-1 sequence can also befused to other types of heterologous sequences, such as a sequence thatencodes another therapeutic gene or a sequence that, when expressed,improves the quantity or quality (e.g., solubility or circulatinghalf-life) of the fusion protein. For example, pGEX vectors can be usedto express the proteins of the invention fused to glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan be easily purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors (Pharmacia Biotech Inc; Smith and Johnson,Gene 67:31-40, 1988) are designed to include thrombin or factor Xaprotease cleavage sites so that the cloned target gene product can bereleased from the GST moiety. Other fusion partners include albumin anda region (e.g., the Fc region) of an immunoglobulin molecule (e.g., IgG,IgA, IgM, or IgE). Other useful vectors include pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.), whichfuse maltose E binding protein and protein A, respectively, to an n-3desaturase.

[0033] Transgene expression can be sufficiently prolonged from episomalsystems, so that readministration of the vector, with its transgene, isnot necessary. Alternatively, the vector can be designed to promoteintegration into the host genome, preferably in a site-specificlocation, which would help ensure that the transgene is not lost duringthe cell's lifetime. Whatever the means of delivery, transcriptionalcontrol, exerted by the host cell, would promote tissue specificity andregulate transgene expression.

[0034] The expression vector will be selected or designed depending on,for example, the type of host cell to be transformed and the level ofprotein expression desired. For example, when the host cells aremammalian cells, the expression vector can include viral regulatoryelements, such as promoters derived from polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40. The nucleic acid inserted (i.e.,the sequence to be expressed; here, fat-1) can also be modified toencode residues that are preferentially utilized in E. coli (Wada etal., Nucleic Acids Res. 20:2111-2118, 1992). These modifications can beachieved by standard recombinant techniques. More generally, theexpression vectors of the invention can be designed to express proteinsin prokaryotic or eukaryotic cells. For example, polypeptides of theinvention can be expressed in bacterial cells (e.g., E. coli), fungi,yeast, or insect cells (e.g., using baculovirus expression vectors). Forexample, a baculovirus such as Autographa californica nuclearpolyhedrosis virus (AcNPV), which grows in Spodoptera frugiperda cells,can be used as a vector to express foreign genes.

[0035] As noted elsewhere, the expression vectors and nucleic acids usedto express fat-1 can also contain a tissue-specific promoter. Suchpromoters are known in the art and include, but are not limited toliver-specific promoters (e.g., albumin; Miyatake et al., 1997),muscle-specific promoters (e.g., myosin light chain 1 (Shi et al., 1997)α-actin), pancreatic-specific promoter (e.g., insulin or glucagonpromoters), neural-specific promoters (e.g., the tyrosine hydroxylasepromoter or the neuron-specific enolase promoter), endothelialcell-specific promoters (e.g., von Willebrandt; Ozaki et al., 1996), andsmooth muscle-cells specific promoters (e.g., 22a; Kim et al., 1997).Tumor-specific promoters are also being used in developing cancertherapies, including tyrosine kinase-specific promoters for B16 melanoma(Diaz et al., 1998), DF3/MUC1 for certain breast cancers (Wen et al.,1993; for breast cancer, an adipose-specific promoter region of humanaromatase cytochrome p450 (p450arom) can also be used (see U.S. Pat. No.5,446,143; Mahendroo et al., J. Biol. Chem. 268:19463-19470, 1993; andSimpson et al., Clin. Chem. 39:317-324, 1993). An a-fetoprotein promotercan be used to direct expression in hepatomas (Chen et al., 1995). Thevectors and other nucleic acid molecules of the invention (e.g., thefat-1 cDNA per se) can also include sequences that limit the temporalexpression of the transgene. For example, the transgene can becontrolled by drug inducible promoters by, for example including cAMPresponse element enhancers in a promoter and treating the transfected orinfected cell with a cAMP modulating drug (Suzuki et al., 1996).Alternatively, repressor elements can prevent transcription in thepresence of the drug (Hu et al., 1997). Spatial control of expressionhas also been achieved by using ionising radiation (radiotherapy) inconjunction with the erg1 gene promoter (Hallaham et al., 1995).Constructs that contain such regulatory sequences are within the scopeof the present invention.

[0036] In the examples that follow, RNA analysis and enzymatic assayswere performed to assess gene expression, and gas chromatography-massspectrometry were used to determine fatty acid profiles (these arestandard techniques that one of ordinary skill in the art could use toassess any variant of the fat-1 sequence for biological activity; orincorporate in any method of assessing a sample obtained from a patientfor fat-1 expression).

[0037] Some of the studies described below were conducted using corticalneurons. Fat-1 expression not only modified the cellular n-6:n-3 fattyacid ratio and eicosanoid profile in these neurons, but also protectedthe cells from apoptosis, thereby increasing cellular viability. Morespecifically, fat-1expression modified the fatty acid ratio andprotected rat cortical neurons against growth factor withdrawal-inducedapoptotis in the absence of supplementation with exogenous n-3 PUFAs.Accordingly, the nucleic acid molecules (and other compositions)described herein can be used as neuroprotectants, which can beadministered to premature infants and to older patients having anyneurodegenerative disease (alternatively, the molecules or othercompositions can be delivered to an animal, parts of which are thenconsumed by the patient). The protective effect of gene transfer onneuronal apoptosis minics the protective effects of n-3 fatty acidsupplementation.

[0038] The positive results obtained with neurons are especiallyencouraging because n-3 PUFA deficiency leads to abnormal development ofthe retina and the brain, particularly in premature infants (Uauy etal., Lipids 36:885-895, 2001), and animals deficient in n-3 PUFA showdeficits in memory, spatial and context-dependent learning, and loss ofvisual acuity (Carrie et al., Neurosci. Lett. 266:69-72, 1999; Yehuda etal., J. Neurosci. Res. 56:565-70, 1999). There are also indications thatvarious neurological disease states in humans are associated with an n-3deficient status (Vancassel et al., Prost. Leuk. Ess. Fatt. Acids65:1-7, 2001; Hoffman and Birch, World Rev. Nutr. Diet 83:52-69, 1998).

[0039] The biological functions of PUFAs are described further here, asthese functions bear on the types of conditions amenable to treatmentwith the nucleic acid molecules (and other compositions) describedherein. PUFAs are important structural components of membranephospholipids and are precursors of families of signaling molecules(eicosanoids) including prostaglandins, thromboxanes, and leukotrienes(Needleman et al., Ann. Rev. Biochem. 55:69-102, 1986; Smith andBorgeat, In Biochemistry of Lipids and Membranes, D. E. Vance & J. E.Vance, Eds., Benjamin/Cummings, Menlo Park, Calif., 00 325-360, 1986).The eicosanoids derived from PUFAs play a key role in modulatinginflammation, cytokine release, the immune response, plateletaggregation, vascular reactivity, thrombosis and allergic phenomena(Dyerberg et al., Lancet 2:117-119, 1978; Cyerberg and Bang, Lancet2:433-435, 1979; James et al., Am. J. Clin Nutr. 7:343S-3438S, 2000;Calder, Ann. Nutr. Metab. 41:203-234, 1997). The principal fatty acidprecursors of these signaling compounds are arachidonic acid (AA,20:4n6), providing an n-6 substrate that is responsible for the majorsynthesis of the series 2 compounds, and eicosapentaenoic acid (EPA,20:5n3), an n-3 substrate that is responsible for the parallel synthesisof many series 3 eicosanoids with an additional double bond. The n-6:n-3ratio in phospholipids modulates the balance between eicosaniods of the2 and 3 series derived from AA and EPA. The eicosanoids derived from AA(series 2) and EPA (series 3) are functionally distinct and some haveimportant opposing physiological functions (Dyerberg et al., Lancet2:117-119, 1978; Cyerberg and Bang, Lancet 2:433435, 1979; James et al.,Am. J. Clin Nutr. 7:343S-3438S, 2000; Calder, Ann. Nutr. Metab.41:203-234, 1997). Series 3 eicosanoids are weak agonists or, in somecases, antagonists of series 2 eicosanoids. For example, eicosanoids ofthe 2 series promote inflammation and platelet aggregation, and activatethe immune resoponse, whereas series 3 eicosanoids tend to amelioratethese effects. In addition, PUFAs, in the form of free fatty acids, areinvolved in gene expression and intercellular cell-to-cell communication(Price et al., Curr. Opin. Lipidol 11:3-7,2000; Sellmayer et al. Lipids31 Suppl:S37-S40, 1996; vonSchacky, J. Lab. Clin. Med. 128:5-6, 1996).Thus, PUFA can exhibit many diverse biological effects.

[0040] The compositions and methods described herein can be used totreat a variety of specific conditions as well as to improve generalhealth. Any condition that is amenable to treatment by administration ofn-3 PUFAs is amenable to treatment by way of the methods of the presentinvention, which comprise administration of a gene encoding an n-3desaturase (e.g., the C. elegans fat-1 gene). Some of the conditionsamenable to treatment are described below.

[0041] n-3 PUFAs have attracted considerable interest as pharmaceuticaland nutraceutical compounds (Connor, Am. J. Clin. Nutr. 70:560S-569S,1999; Simopoulos, Am. J. Clin. Nutr. 70:562S-569S, 1999; Salem et al.,Lipids 31:S1-S326, 1996). During the past 25 years, more than 4,500studies have explored the effects of n-3 fatty acids on human metabolismand health (e.g., cardiovascular health). From epidemiology to cellculture and animal studies to randomized controlled trials, thecardioprotective effects of omega-3 fatty acids have been recognized(Leaf and Kang, World Rev. Nutr. Diet. 83:24-37, 1998; De Caterina etal., Eds., n-3 Fatty Acids and Vascular Disease, Springer-Verlag,London, 1999, pp 166; O'Keefe and Harris, Mayo Clin. Proc. 75:607-614,2000). The predominant beneficial effects include a reduction in suddendeath (Albert et al., JAMA 279:23-28, 1998; Siscovick et al., JAMA274:1363-1367, 1995), decreased risk of arrhythmia (Kang and Leaf,Circulation 94:1774-1780, 1996), lower plasma triglyceride levels(Harris, Am. J. Clin. Nutr. 65:1645S-1654S, 1997), and a reducedblood-clotting tendency (Agren et al., Prostagland. Leukot. Esseizt.Fatty Acids 57:419-421, 1997; Mori et al., Arterioscler. Throm. Basc.Biol. 17:279-286, 1997). Evidence from epidemiological studies showsthat another n-3 fatty acid, α-linolenic acid, reduces risk ofmyocardial infarction (Guallar et al., Arterioscler. Thromb. Vasc. BioL19:1111-1118, 1999) and fatal ischemic heart disease in women (Hu etal., Am. J. Clin. Nutr. 69:890-897, 1999). Several randomized controlledtrials recently have demonstrated beneficial effects of both α-linolenicacid (de Lorgeril et al., Circulation 99:779-785, 1999) and marine ω-3fatty acids (Singh et al., Cardiovasc. drugs ther. 11:485-491, 1997; VonSchacky et al., Ann. Intern. Med. 130:554-562, 1999; GISSI-PrevenzioneInvestigators, Lancet 354:447-455, 1999) on both coronary morbidity andmortality in patients with coronary disease. The n-3 fatty acid, EPA,exerts anticancer activity in vitro and in animal models of experimentalcancer (Bougnoux, Curr. Opin. Clin. Nutr. Metab. Care 2:121-126, 1999;Cave, Breast Cancer Res. Treat. 46:239-246, 1997). Human studies showthat populations whose diets are rich in EPA exhibit a remarkably lowincidence of cancer (Rose and Connolly, Pharmacol. Ther. 83:217-244,1999). Supplementation with n-3 PUFAs shows therapeutic effects oninflammatory and autoimmune diseases such as arthritis (Kremer, Am. J.Clin. Nutr. 71:349S-351 S, 2000; Ariza-Ariza et al., Semin. ArthritisRheum. 27:366-370, 1998; James et al., Am. J. Clin. Nutr. 71:343S-348S),and studies with nonhuman primates (Neuringer et al., Proc. Natl. Acad.Sci. USA 83:4021-4025, 1986) and human newborns (Uauy et al., Proc.Nutr. Soc. 59:3-15, 2000; Uauy et al., Lipids 31:S167-176, 1996)indicate that the n-3 fatty acid, DHA, is essential for the normalfimctional development of the retina and brain, particularly inpremature infants. Furthermore, n-3 PUFA have been shown to havebeneficial effects on many other clinical problems, such as hypertension(Appel et al., Arch. Intern. Med. 153:1429-1438, 1993), diabetes (Rahejaet al., Ann. N.Y. Acad. Sci. 683:258-271, 1993), obesity (Clarke, Br. J.Nutr. 83:S59-66, 2000), skin disorders (Ziboh, World Rev. Nutr. Diet.66:425435, 1991), renal disease (De Caterina et al., Kidney Int.44:843-850, 1993), ulcerative colitis (Stenson et al., Ann. Intern. Med.116:609-614, 1992), Crohn's disease (Belluzzi et al., N. Engl. J. Med.334:1557-1560, 1996), chronic obstructive pulmonary disease (Shahar etal., N. Engl. J. Med. 331:228-233, 1994), and transplanted organrejection (Otto et al., Transplantation 50:193-198, 1990). In general, abalanced n-6:n-3 ratio of the body lipids is essential for normal growthand development and plays an important role in the prevention andtreatment of many clinical problems. The diseases, disorders, andconditions described above are amenable to treatment with the nucleicacid molecules (and other compositions) described herein.

[0042] According to recent studies (Simopoulos, Poultry Science79:961-970, 2000), the ratio of n-6 to n-3 essential fatty acids intoday's diet is around 10-20:1. This indicates that present Westerndiets are deficient in n-3 fatty acids compared with the diet on whichhumans evolved and their genetic patterns were established (n-6/n-3=1:1)(Leaf and Weber, Am. J. Clin Nutr. 45:1048-1053, 1987). Since the n-6and n-3 fatty acids are metabolically and functionally distinct and haveimportant opposing physiological functions, their balance is importantfor homeostasis and normal development. However, n-3 and n-6 PUFAs arenot interconvertible in the human body because mammalian cells lack theenzyme n-3 desaturase. Therefore, the balance between n-6 and n-3 PUFAin biological membranes is regulated based on dietary supply. Elevatingthe tissue concentrations of n-3 fatty acids in human subjects oranimals relies on increased consumption of n-3 PUFA-enriched foods orn-3 PUFA supplements. Given the potential therapeutic actions of n-3PUFAs, an international scientific working group has recommended dietsin which the intake of n-6 fatty acids is decreased and the intake ofn-3 fatty acids is increased (Simopoulos, Food Australia 51:332-333,1999). The American Heart Association has also recently made such adietary recommendation (AHA Dietary Guidelines: Revision 2000,Circulation 102:2284-2299, 2000).

[0043] Although dietary supplementation with n-3 PUFA is a safeintervention, it has a number of limitations. For example, to achieve asignificant increase in tissue concentrations of n-3 PUFA in vivorequires a chronic intake of high doses of n-3 PUFA for a period of atleast 2-3 months. Bioavailability of fatty acids to cells from the dietinvolves a series of physiological processes including digestion,absorption, transport and metabolism of fat. Thus, the efficacy ofdietary intervention depends on the physiological and health status ofan individual. A patient in critical condition or who has agastrointestinal disorder is unlikely to be able to ingest or absorbfatty foods or n-3 PUFA supplements. In addition, encapsulated fish oilsupplements are unlikely to be suited to daily use over a person'slifetime because of their high caloric content. Moreover, ingestion ofsome species of fish from costal waters and lakes may carry toxicamounts of mercury or organic toxins, and effective dietary interventionrequires a disciplined change in dietary habits that some people may notbe able to sustain. In view of the foregoing, there is a great need forthe means to quickly and effectively increase cellular n-3 PUFA contentand balance the n-6:n-3 ratio without resorting to long-term intake offish or fish oil supplements. This need is met by the methods of thepresent invention, which create an alternative food source (viatransgenic livestock whose cells contain substantially more n-3 PUFAsthan in non-transgenic animals) or which provide for administration of agene encoding an n-3 desaturase enzyme to patients (e.g., humanpatients). A particular advantage of the present methods is that theynot only elevate tissue concentrations of n-3 PUFAs, but alsosimultaneously decreases the levels of excessive endogenous n-6 PUFA.

EXAMPLES Example 1 Construction of a Recombinant Adenovirus

[0044] A recombinant adenovirus carrying the fat-1 gene was constructedfollowing procedures similar to those described by He et al. (Proc.Natl. Acad. Sci. USA 95:2509-2514, 1998). The n-3 fatty acid desaturasecDNA (fat-1 gene) in pCE8 was kindly provided by Dr. J. Browse(Washington State University) (but can be synthesized or cloned usinginformation and techniques available to those of ordinary skill in theart; see Spychalla et al., Proc. Natl. Acad. Sci. USA 94:1142-1147,1997; U.S. Pat. No. 6,194,167; and FIGS. 17A and 17B). The cDNA insertof pCE8 was excised from the plasmid with an EcoRI/KpnI double digest,inserted into a shutter vector, and then recombined with an adenoviralbackbone according to the methods of He et al. (supra). Two,first-generation type 5 recombinant adenoviruses were generated: Ad.GFP,which carries the green fluorescent protein (GFP, as reporter gene)under control of the cytomegalvirus (CMV) promoter, and Ad.GFP.fat-1,which carries both the fat-1 and GFP genes, each under the control ofseparate CMV promoters. The recombinant viruses were prepared as hightiter stocks through propagation in 293 cells, as described previously(Hajjar et al. Circulation 95:423-429, 1997). The constructs wereconfirmed by enzymatic digestion and by DNA sequence analysis. See alsoHajjar et al., Circulation 95:4230429, 1997 and Hajjar et al., Circ.Res. 81:145-153, 1997.

[0045] Wild-type adenovirus contamination can be assessed and shown tobe excluded by the absence of both PCR-detectable E1 sequences andcytopathic effects on the nonpermissive A549 cell line. Alternativeadenoviral vectors with other promoters or adeno-associated viral (AAV)vectors can be constructed if necessary or desired.

Example 2 Culture and Infection of Cardiac Myocytes with Adenovirus

[0046] Cardiac myocytes were isolated from one-day-old rats using theNational Cardiomyocyte Isolation System (Worthington Biochemical Corp.,Freehold, N.J.). The isolated cells were placed in 6-well plates andcultured in F-10 medium containing 5% fetal bovine serum and 10% horseserum at 37° C. in a tissue culture incubator with 5% CO₂ and 98%relative humidity. Cells were used for experiments after 2-3 days ofculture. Viral infection was carried out by adding viral particles atdifferent concentrations (5×10⁹-10¹⁰ pfu) to culture medium containing2% fetal bovine serum (FBS). After a 24 hour incubation, the infectionmedium was replaced with normal (15% serum), culture medium supplementedwith 10 μM of 18:2n-6 and 20:4n-6. About 48 hours after infection, thecells can be used (e.g., one can then analyze gene expression, fattyacid composition, viability, or growth (e.g., proliferation or rate ofdivision)).

Example 3 Detecting Fat-1 Expression with Fluorescence Microscopy andRNA Analysis

[0047] Gene expression can be assessed by many methods known in the artof molecular biology. Here, expression of fat-1 in cardiac myocytes,infected as described above, was assessed by visual examination ofinfected cells and a ribonuclease (RNase) protection assay.

[0048] More specifically, the coexpression of GFP allowed us to identifythe cells that were infected and expressed the transgene. About 48 hoursafter infection, almost all of the cells (>90%) exhibited brightfluorescence, indicating a high efficiency of gene transfer and a highexpression level of the transgene (see FIG. 1). Expression of fat-1transcripts was also determined by RNase protection assay using a RPAIII™ it (Ambion). Briefly, total RNA was extracted from cultured cellsusing an RNA isolation kit (Qiagen) according to the manufacturer'sprotocol. The plasmid containing the fat-1 gene, pCE8, was linearizedand used as a transcription template. Anti-sense RNA probes weretranscribed in vitro using ³³P-UTP, hybridized with the total RNAextracted from the myocytes, and digested with RNase to removenon-hybridized RNA and probe. The protected RNA:RNA was resolved byelectrophoresis through a denaturing gel and subjected toautoradiography. A probe targeting the β-actin gene was used as acontrol. Fat-1 mRNA was not detected in cells infected with AD.GFP (alsoused as a control), but was abundant in cells infected with Ad.GFP.fat-1(FIG. 2). This result indicates that adenovirus-mediated gene transferconfers very high expression of fat-1 gene in rat cardiac myocytes thatnormally lack the gene.

Example 4 Lipid Analysis; The Effect of n-3 Sesaturase on Fatty AcidComposition

[0049] By lipid analysis, one can determine whether the expression of afat-1 gene in cardiac myocytes (or any other cell type) converts n-6fatty acids to n-3 fatty acids and, thereby, changes the fatty acidcomposition of the cell. Following infection with the adenovirusesdescribed above, cells were incubated in medium supplemented with n-6fatty acids (10 μM 18:2n-6 and 10 μM 20:4n-6) for 2-3 days. After theincubation, the fatty acid composition of total cellular lipids wasanalyzed as described previously (Kang et al., Biochim. Biophys. Acta.1128:267-274, 1992; Weylandt et al., Lipids 31:977-982, 1996).

[0050] Lipid was extracted with chloroform/methanol (2:1, v/v)containing 0.005% butylated hydroxytoluene (as antioxidant). Fatty acidmethyl esters were prepared using 14% BF3/methanol reagent. Fatty acidmethyl esters are quantified by GC/MS using a HP5890 Series II gaschromatograph equipped with a Supelcowax SP-10 capillary column attachedto a HP-5971 mass spectrometer. The injector and detector are maintainedat 260° C. and 280° C., respectively. The oven program is initiallymaintained at 150° C. for 2 minutes, then ramped to 200° C. at 10°C./min and held for 4 minutes, ramped again at 5° C./min to 240° C.,held for 3 minutes, and finally ramped to 270° C. at 10° C./min andmaintained for 5 minutes. Carrier gas flow rate is maintained at aconstant 0.8 mL/min throughout. Total ion monitoring is performed,encompassing mass ranges from 50-550 amus. Fatty acid mass is determinedby comparing areas of various analyzed fatty acids to that of a fixedconcentration of internal standard.

[0051] The fatty acid profiles were remarkably different between thecontrol cells infected with Ad.GFP and the cells infected withAd.GFP.fat-1 (FIG. 3). Moreover, cells infected with Ad.GFP showed nochange in their fatty acid profiles when compared with non-infectedcells. In the cells expressing the fat-1 gene (n-3 desaturase), almostall kinds of n-6 fatty acids were largely converted to the correspondingn-3 fatty acids, namely, 18:2n-6 to 18:3n-3, 20:2n-6 to 20:3n-3, 20:3n-6to 20:4:n-3, 20:4n-6 to 20:5n-3, and 22:4n-6 to 22:5n-3. As a result,the fatty acid composition of the cells expressing fat-1 wassignificantly changed with respect to that of the control cells infectedwith Ad.GFP (FIG. 5). Importantly, the ratio of n-6:n-3 was reduced from15:1 in the control cells to 1:1.2 in the cells expressing the n-3 fattyacid desaturase.

Example 5 Measuring Eicosanoids Following Fat-1 Expression

[0052] Since 20:4n-6 (AA) and 20:5n-3 (EPA) are the precursors of2-series and 3-series of eicosanoids, respectively, differences in thecontents of AA and EPA may lead to a difference in production ofeicosanoids in the cells. Thus, we measured the production ofeicosanoids in the infected cells following stimulation with calciumionophore A23187 by using a EIA kit that specifically detectprostaglandin E₂ with a 16% cross-reactivity with prostaglandin E3. Morespecifically, Prostaglandin E₂ was measured by using enzyme immunoassaykits (Assay Designs, Inc) following the manufacturer's protocol. (Thecross-reactivity with PGE3 is 16%). Cultured cells were washed andserum-free medium containing calcium ionophore A23187 (5 μM). After a 10minute incubation, the conditioned medium was recovered and subjected toeicosanoid measurement. The amount of prostaglandin E₂ produced by thecontrol cells was significantly higher than that produced by cellsexpressing the n-3 desaturase encoded by fat-1 (FIG. 4).

Example 6 Analysis of Animal Cells in Culture

[0053] In this example and the two that follow, we set out threedifferent experimental models: cultured cells (other types of culturedcells are tested further below), adult rats, and transgenic mice. Asshown above, the cultured cell model can be used to characterize theenzymatic properties and biochemical effects of the n-3 desaturase whenexpressed in mammalian cells in vitro; the adult rat model can be usedto evaluate the efficacy with which a transferred fat-1 gene can elevatetissue concentrations of n-3 PUFA in vivo, and the transgenic mousemodel can be used to assess the long-term and systematic effects of thetransgene on lipid composition of various tissues or organs in vivo. Forthe first two models, the introduction of the fat-1 gene into mammaliancells/tissues will be carried out by mean of adenoviral gene transfer(mediated by recombinant adenoviruses). For the last model, genetransfer will be carried out by microinjection of the transgene intofertilized mouse eggs. Following gene transfer, the expression profileof the transferred gene can be characterized by mRNA and/or proteinanalysis (see, e.g., Example 3, above), and the biochemical effects,mainly the fatty acid composition of the cells or tissues, will bedetermined by GC-MS technology (see, e.g., Example 4, above).Eicosanoids will be measured by enzyme immunoassay (see, e.g., Example5). Changes are identified by comparing the data obtained fromfat-1-expressing cells with data obtained from control cells or tissuesinfected with the same (or a similar) virus, but not transfected withfat-1. The end point of these studies is the biochemical changes incellular fatty acid composition and eicosanoid profile.

[0054] Cultures of virtually any animal cells (including human celllines) can be infected with recombinant adenovirus (Ad.GFP.fat-1 orAd.GFP), after which expression of the transferred gene can be assessedby RNA or protein analysis. The experimental procedures and relatedmethods are described in the Examples above and outlined in FIG. 6.Various cell types including cardiac myocytes, neurons, hepatocytes,endothelial cells, and macrophages have been used in studies of n-3fatty acids.

[0055] Cardiac myocytes can be isolated and cultured as described above(see Example 2), and other cell types, such as cerebellar granuleneurons and hepatocytes can be prepared from 1-5 day-old rats followingthe method described by Schousboe et al. (In A Dissection and TissueCulture Manual of the Nervous System, Shahar et al., Eds., Alan R. Liss,New York, N.Y., pp. 203-206, 1989). Human cell lines, including breastcancer cell lines and leukemia cell lines can be cultured in MEN mediumor RPMI 1640 supplemented with 10% fetal bovine serum (FBS) in a 37°C./5% CO₂ incubator.

[0056] Viral infection can be carried out by adding viral particles atvarious concentrations (e.g., 2×10⁹-2×10¹⁰ pfu) to culture mediumcontaining no FBS or 2% FBS (see also Example 2). ARer a 24-hourincubation, the infection medium is replaced with normal (10% FBS)culture medium. Forty-eight hours after infection, cells can be used foranalysis of gene expression or fatty acid composition. Transgeneexpression can be assessed by fluorescence microscopy when a fluorescenttag is included in the transgene (see Example 1 and FIG. 1; similarly,the tag can be an antigenic protein detected by a fluorescent antibody)or by a standard RNA assay (e.g. a Northern blot or RNase protectionassay). Since the fat-1 gene normally does not exist in control cells,it is not difficult to identify the difference in fat-1 mRNA between thecontrol cells and cells expressing fat-1.

[0057] n-3 desaturase catalyzes the introduction of an n-3 double bondinto n-6 fatty acids, leading to formation of n-3 fatty acids with onemore double bond than their precursor n-6 fatty acids (e.g.,18:2n-6→18:3n-3, 20:4n-6-20:5n-3). The rate of conversion of substratesto products (the amount of products formed within a given time period)is thought to be directly proportional to the expression/activity of adesaturase. Thus, the fimctional activity of this enzyme can bedetermined, from a sample obtained from an animal (e.g., a tissuesample) or in cultured cells by measurement of the conversions (thequantity of products) using the following methods.

[0058] Fatty acid desaturation assay using radiolabeled n-6fatty acidsas substrates: The assay can be performed following the protocoldescribed by Kang et al. (Biochim. Biophys Acta. 1128:267-274, 1992).Briefly, various labeled n-6 fatty acids (e.g., [¹⁴C]18:2n-6,[¹⁴C]20:4n-6) bound to BSA are added to serum-free culture medium andincubated with cells for 4-6 hours. After that, cells and culture mediumwill be harvested. Lipids are extracted and methylated (see below). Thelabeled fatty acid methyl esters are separated according to degree ofunsaturation (i.e., the number of double bond) on silica-gel TLC platesimpregnated with AgNO₃. Bands containing fatty acids with differentdouble bonds can be identified by comparison with reference standards.Quantity of the labeled fatty acids is determined by scintillationcounting, and data are compared between control cells and the cellsexpressing the fat-1 gene.

[0059] Fatty acid analysis by gas chromatography: Conversion of fattyacids can be determined more accurately by analysis of fatty acidcomposition using gas chromatography-mass spectrometry (see below).Using this method, no radiolabeled fatty acid is required. Fatty acidcontents of cultured cells expressing the n-3 desaturase gene, in thepresence of various substrates, can be analyzed. The conversion of eachfatty acid can be determined by comparison of fatty acid profilesbetween control cells and the cells expressing the fat-1 gene.

[0060] The fatty acid composition of total cellular lipids orphospholipids can be analyzed as described previously (Kang et al.,Biochim. Biophys. Acta. 1128:267-274, 1992; Weylandt et al., Lipids31:977-982, 1996). The procedures are as follows:

[0061] Lipid extraction (see also Example 4): Five ml ofchloroform/methanol (2:1, v/v) containing 0.005% butylatedhydroxytoluene (as antioxidant) is added to washed cell pellets andvortexed vigorously for 1 minute then left at 4° C. overnight. One ml of0.88% NaCl is added and mixed again. The chloroform phase containinglipids is collected. The remains are extracted once again with 2 mlchloroform. The chloroform is pooled and dried under nitrogen and storedin sealed tubes at −70° C.

[0062] Separation of lipids by thin-layer chromatography (TLC): TLCplates are activated at 100° C. for 60 minutes. TLC tanks areequilibrated with solvent for at least one hour prior to use. Totalphospholipid and triacyglycerol are separated by running the sample onsilica-gel G plates using a solvent system comprised of petroleumether/diethyl ether/acetic acid (80:20:1 by vol.) for 30-35 minutes.Individual phospholipids are separated by TLC on silica-gel H platesusing the following solvent system: chloroform/methanol/2-propanol/0.25%KCl/triethylamine (30:9:25:6:18 by vol.). Bands containing lipids aremade visible with 0.01% 8-anilino-1-naphthalenesulfonic acid, and gelscrapings of each lipid fraction are collected for methylation.

[0063] Fatty acid methylation: Fatty acid methyl esters are preparedusing 14% BF₃/methanol reagent. One or two ml of hexane and 1 ml ofBF₃/methanol reagent are added to lipid samples in glass tubes withTeflon-lined caps. After being flushed with nitrogen, samples are heatedat 100° C. for one hour, cooled to room temperature and methyl estersare extracted in the hexane phase following addition of 1 ml H₂O.Samples are allowed to stand for 20-30 minutes, the upper hexane layeris removed and concentrated under nitrogen for GC analysis.

[0064] Gas chromatography-mass spectrometry. Methylated samples arereconstituted in 100-200 μl hexane or isooctane of which 1-2 μl will beanalyzed by gas chromatography. An Omegawas column (30 m; Supelco,Bellefonte, Pa.) will be used in a Hewlett-Packard 5890A gaschromatograph (Hewlett-Packard, Avondage, Pa.). Carrier gas is hydrogen(2.39 ml/min), injected with a split ratio of 1:31. The temperature isinitially 165° C. for 5 minutes, then is increased to 195° C. at 2.5°C./min and, from there, to 220° C. at 5° C./min. The temperature is heldfor 10.5 minutes and then decreased to 165° C. at 27.5° C./min. Peakswill be identified by comparison with fatty acid standards(Nu-Chek-Prep, Elysian, Minn.), and area percentage for all resolvedpeaks will be analyzed using a Perkin-Elmer M1 integrato (Perkin-Elmer,Norwood, Conn.). These analytical conditions separates all saturated,mono, di- and polyunsaturated fatty acids from C14 to C25 carbons inchain length. The sample size will be calculated based on externalstandards when added. In addition, the gas chromatography-massspectrometry (GC-MS) will be carried out using a Hewlett-Packard massselective detector (model 5972) operating at an ionization voltage of 70eV with a scan range of 20-500 Da. The mass spectrum of any new peakobtained will be compared with that of standards (Nu Chek Prep, Elysian,Minn.) in the database NBS75K.L (National Bureau of Standards).

Example 7 Evaluation of n-3 Sesaturase Gene Transfer In Vivo

[0065] The experiments described here allow introduction of the fat-1gene into aniimal tissues or organs (e.g., heart), where the enzymeproduct can quickly optimize fatty acid profiles by increasing thecontent of n-3 PUFAs and decreasing the content of n-6 PUFAs. The heartis selected as an experimental target for the gene transfer because ithas been well studied in relation to n-3 fatty acids, and it is a vitalorgan.

[0066] Adult rats, fed a normal diet or a diet high in n-6 PUFA for twomonths, will be randomized to receive either an adenovirus carrying thefat-1 gene (Ad.GFP.fat-1) or an adenovirus carrying the reporter geneGFP (Ad. GFP, as control). The adenoviruses will be delivered to theheart of a living animal using a catheter-based technique, which canproduce an expression pattern that is grossly homogeneous throughout theheart (Hajjar et al., Proc. Natl. Acad. Sci. USA 95:525105256, 1998).Two days, 4 days, 10 days, 30 days and 60 days after infection (genetransfer), animals will be sacrificed, and their hearts will beharvested and used for determination of the transgene expression andanalysis of fatty acid composition. Another group of rats will be fed adiet rich in n-3 fatty acids (low n-6/n-3 ratio) for two months withoutgene transfer and used as references. These experiments (in whichanimals are on different diets and samples harvested at different timepoints) are designed to determine whether transfer of the fat-1 gene canbring about a desired biochemical effect (n-6/n-3 ratio, eicosanoidprofile) similar to or even superior to that induced by dietaryintervention (i.e., n-3 FA supplementation), how quickly a significantchange in fatty acid composition can be achieved, and how long thechange can last. Rats injected with the reporter (GFP) gene will be usedas controls (our preliminary studies showed that gene transfer of GFPhas no effect on fatty acid composition). The experimental flow chart isshown in FIG. 7.

[0067] Animals and Diets: weight-matched adult Sprague-Dawley rats willbe randomly assigned to three groups. Each group is fed with one ofthree different diets: normal (basal) diet, a high n-6 diet, or a highn-3 diet. These diets are prepared as follows.

[0068] Basal diet: a commercial rat fat-free diet (Agway Inc. C.G.,Syracuse, N.Y.) to which 2% (w/w) corn oil is added; High n-6 diet: thebasal diet supplemented by addition of a further 13% (w/w) corn oil orsafflower oil (high in n-6 fatty acids), bringing the final diet to atotal of 15% fat; High n-3 diet: the basal diet supplemented with 13%(w/w) fish oil (30% EPA, 20% DHA, 65% total n-3 PUFA) (Pronova BiocareA/S, Oslo), bringing the final diet to a total of 15% fat. This groupwill serve as a control group for this study.

[0069] The diets will be prepared in small batches weeldy, kept at −20°C. and thawed daily in the amounts required. Vitamin E (100 mg/100 gfat) and butylated hydroxy toluene (final concentration 0.05%) will beadded to prevent oxidation of long-chain polyunsaturated fatty acids(The BHT should serve to prevent autooxidation of the unsaturated fattyacids during preparation and storage). To ensure animals are receivingadequate nutrition, the rats in all groups will be weighted weekly.After 8 weeks on the diets, the animals will be subjected to genetransfer.

[0070] Adenoviral Delivery Protocol. The delivery of adenoviruses to theheart will be performed by using a cathether-based technique similar tothat described by Hajjar et al (supra). Briefly, rats will beanesthetized with intra peritoneal pentobarbital (60 mg/kg) and placedon a ventilator. The chest is entered from the left side through thethird intercostals space. The pericardium is opened and a 7-0 sutureplaced at the apex of the left ventricle. The aorta and pulmonary arteryare identified. A 22-gauge catheter containing 200 μL adenovirus(9-10×10¹⁰ pfu/ml) is advanced from the apex of the left ventricle (LV)to the aortic root. The aorta and pulmonary arteries are clamped distalto the site of the catheter, and the solution is injected. The clamp ismaintained for 10 seconds while the heart pumped against a closed system(isovolumically). After 10 seconds, the clamp on the aorta and pulmonaryartery is released, the chest is closed, and the animals are extubatedand transferred back to their cages.

[0071] At day 2, 4, 10, 30 and 60 after gene transfer, animals will besacrificed, their hearts infected with the viruses will be removed,perfused or rinsed with saline to removed all blood and a portion of thetissues will be promptly frozen at −80° C. for lipid analysis andeicosanoid measurement. The remaining tissues will be used fordetermination of the mRNA levels and/or protein levels of the n-3desaturase.

[0072] It is possible that other organs such as brain and liver may alsobe infected at high levels by the adenoviruses entering the bloodstream. Thus, other organs, in addition to the heart, will be alsoharvested for analyses of transgene expression and lipid profile.

[0073] Other methods, including assessment of transgene expression (byNorthern blot, RNase protection assay, or in situ hybridization),analysis of fatty acid composition, measurement of eicosanoids, andstatistical analysis will be carried out, as described above in thecontext of cultured cells.

Example 8 Transgenic Animals

[0074] The studies described here are designed to create transgenic micethat globally express the fat-1 gene and to characterize the tissue andorgan lipid profiles of these animals. Transgenic mice have become avaluable model for evaluation of physiological significance of a gene invivo. Availability of transgenic mice allows us to study the effect of atransgene in a variety of cell types at different stages of an animal'slifespan. This n-3 transgenic mouse model will provide new opportunitiesto elucidate the roles of n-3 PUFA and compounds derived from them inthe development and cell biology.

[0075] To generate transgenic animals that can globally express thefat-1 gene, one can use an expression vector that contains a fat-1 geneand the chicken beta-actin promotor with the CMV enhancer (CAGpromotor), which is highly active in a wide range of cell types andtherefore allows high-level and broad expression of the transgene (Niwaet al., Gene 108:193-199, 1991; Okabe et al., FEBS Lett. 407:313-319,1997). The expression construct will be microinjected into the pronucleiof one-cell embryos of C57BL/6X C3H mice to produce transgenic mice.They will be bred and transgenic mouse line is established. Weanlingmice are fed either a normal diet or a diet high in n-6 PUFA. Varioustissues will be harvested from these animals at different ages (neonate,wean—1 month, adult—6 ms and aging—12 ms, 3-5 mice per time point willbe used) for assessment of the expression levels of the transgene anddetermination of fatty acid composition. The levels of eicosanoids inplasma and various tissues will also be measured. A group of wild-typemice (C57BL/6) fed with the same diet (either a normal diet or a highn-6 diet) will be used as controls. The results will be compared withthose from wild type animals fed the same diet. The procedure isillustrated in FIG. 8.

[0076] The transgene will be prepared by methods similar to thosedescribed by Okabe et al. (supra). Briefly, a cDNA encoding the fat-1gene is amplified by PCR with primers, 5-agaattcggcacgagccaagtttgaggt-3′ (SEQ ID NO: 1) and 5′-gcctgaggctttatgcattcaacgcact-3′ (SEQID NO:2), using pCE8-fat1 (provided by Dr. J. Browse, Washington StateUniversity) as a template. No additional amino acid sequence is added oneither side of the fat-1. The PCR product will be confirmed by DNAsequencing. The EcoR1 and Bgl-II sites included in the PCR primers areused to introduce the amplified fat-1 cDNA into a pCAGGS expressionvector containing the chicken beta-actin promoter and cytomegalovirusenhancer, beta-actin intron and bovine globin poly-adenylation signal(provided by Dr. J Miyazaki, Osaka University Medical School). Theentire insert with the promoter and coding sequence will be excised withBamHI and Sal1 and gel-purified.

[0077] Transgenic mouse lines will be produced by injecting the purifiedBamHI and SalI fragment into C57BL/6 X C3H fertilized eggs. TheDNA-injected eggs are transplanted to pseudo-pregnant mice (B6C3F1) toproduce transgenic mice. The founder transgenic mice will be identifiedby PCR and Southern blot analyses of tail DNA and bred with C57BL/6Jmice. Offspring (either heterozygote or homozygote) will be useddependent on the expression levels of the transgene or phenotype.

[0078] Weanling transgenic mice will be fed either a normal diet or adiet high in n-6 PUFA (see above). Animals will be sacrificed atdifferent ages (neonate, wean to 1 month, adult to 6 mos and aging—12mos, 3-5 mice per time point will be used) and various tissues will beharvested for assessment of the expression level of transgene anddetermination of fatty acid composition. The results will be comparedwith those from wild type animals fed the same diet.

[0079] Other methods, including assessment of transgene expression(Northem blot, RNase protection assays, or in situ hybridization),analysis of fatty acid composition, measurement of eicosanoids, andstatistical analyses will be carried out as described above.

Example 9 Inhibition of Neuronal Cell Death

[0080] Construction of Recombinant Adenovirus (Ad): A recombinant Adcarying the fat-1 gene was constructed as described previously (Kang etal., Proc. Natl. Acad. Sci. USA 98;4050-4054, 2001; see also, above).The n-3 fatty acid desaturase cDNA (fat-1 gene) used was that describedabove, provided in plasmid pCE8. The fat-1 cDNA was excised from theplasmid with an EcoRI/KpnI digestion, and inserted into pAdTrack-CMVvector. The construct was subsequently recombined homologously with anadenoviral backbone vector (pAdEasy 1) to generate two clones: Ad-GFP,which expresses GFP as a reporter or marker, and Ad-GFP-fat-1, whichcarries both the fat-1 and the GFP genes, each under the control ofseparate CMV promoters. Recombinant adenoviral vector DNA was digestedwith PacI. The linerized vector DNA was mixed with SuperFect™ (QIAGEN)and used to infect 293 cells. The recombinant viruses were prepared ashigh-titer stocks through propagation in 293 cells. The integrity of theconstructs was confirmed by enzymatic digestion (i.e., restrictionmapping) and by DNA sequencing. Purified virus was checked and itssequence confirmed again by PCR analysis.

[0081] Tissue Culture and Infection with Ad: Rat cortical neurons wereprepared using standard techniques. Briefly, prenatal embryonic day 17(E17) rat cortical neurons were dissociated and plated inpoly-lysine-coated wells at 2×10⁶ cells/well. The cells were grown inNeurobasal™ Medium (NBM, Life Technologies) supplemented with 25 μMglutamic acid (Sigma Chemical Co., St. Louis, Mo.), 0.5 mM glutamine, 1%antibiotic-antimycotic solution, and 2% B27 (Life Technologies).Cultures were kept at 37° C. in air with 5% CO₂ and 98% relativehumidity. The culture medium was changed every four days. After 8-10days in culture, cells were transfected with either the Ad-GFP (control)or the Ad-GFP-fat-1 plasmids. Viral infections were carried out byadding viral particles to the culture medium. After a 48-hourincubation, cells were used for analyses of gene expression, fatty acidcomposition, eicosanoid production, and induction of apoptosis.

[0082] RNA Analysis: The level of fat-1 expression was determined byprobing for mRNA transcripts in an RNAse protection assay using the RPAIII™ kit (Ambion, Austin, Tex.). Briefly, total RNA was extracted fromcultured cells using a total RNA isolation reagent (TRizol, GIBco BRL)according to the manufacturer's protocol. The plasmid containing thefat-1 gene, pCE8, was linearized and used as a transcription template.Antisense RNA probes were transcribed in vitro using [³³P]-UTP, T7polymerase (Riboprobe System™ T7 kit, Promega), hybridized with totalRNA (15 μg) extracted from neurons, and digested with ribonuclease toremove nonhybridized RNA and probe. The protected RNA-RNA hybrids wereresolved in a denaturing 5% sequence gel and subjected toautoradiography. A probe targeting the β-actin gene was used as aninternal control. fat-1 mRNA was not detected in cells infected withAd-GFP (control), but was highly abundant in cells infected withAd-GFP-fat-1.

[0083] The cells were also examined by fluoresence microscopy. Infectedcells that expressed the fat-1 gene were readily identifiable becausethey co-expressed GFP. Forty-eight hours after infection, 30-40% of theneurons were infected and expressed GFP. These results demonstrate thatAd-mediated gene transfer confers high expression of fat-1 gene in ratcortical neurons, which normally lack the gene.

[0084] Lipid Analysis: The fatty acid composition of total cellularlipids was analyzed as described in Kang et al. (Proc. Natl. Acad. Sci.USA 98:4050-4054, 2001). Lipid was extracted with chloroform:methanol(2:1, vol:vol) containing 0.005% butylated hydroxytoluene (BHT, as anantioxidant). Fatty acid methyl esters were prepared using a 14%(wt/vol) BF3/methanol reagent. Fatty acid methyl esters were quantifiedwith GC/MS by using an HP-5890 Series II gas chromatograph equipped witha Supelcowax™ SP-10 capillary column (Supelco, Bellefonte, Pa.) attachedto an HP-5971 mass spectrometer. The injector and detector aremaintained at 260° C. and 280° C., respectively. The oven program ismaintained initially at 150° C. for 2 minutes, then ramped to 200° C. at10° C./minute and held for 4 minutes, ramped again at 5° C./minute to240° C., held for 3 minutes, and finally ramped to 270° C. at 10°C./minute and maintained for 5 minutes. Carrier gas-flow rate ismaintained at a constant 0.8 ml/min throughout. Total ion monitoring isperformed, encompassing mass ranges from 50-550 atomic mass units. Fattyacid mass is determined by comparing areas of various analyzed fattyacids to that of a fixed concentration of internal standard.

[0085] The expression of fat-1 resulted in conversion of n-6 fatty acidsto n-3 fatty acids, and thus a significant change in the ratio ofn-6:n-3 fatty acids. The fatty acid profile obtained from control cellsis significantly different from that of cells infected with Ad-GFP-fat-1(FIG. 9; see also FIG. 10). Cells infected with Ad-GFP show no change infatty acid composition when compared with non-infected cells. In cellsexpressing the n-3 desaturase, almost all types of n-6 fatty acids wereconverted to the corresponding n-3 fatty acids, namely, 18:2n-6 to18:3n-3, 20:4n-6 to 20:5n-3, 22:4n-6 to 22:5n-3, and 22:5n-6 to 22:6n-3.The change in fatty acid composition of the cells expressing the fat-1gene resulted in reduction of the n-6:n-3 ratio from 6.4:1 in thecontrol cells to 1.7:1 in the cells expressing the n-3 desaturase.Expression of the C. elegans n-3 fatty acid desaturase resulted in asignificant increase in the levels of DHA in transfected cells. Anincrease in levels of EPA and ALA is observed with a concomitantdecrease in AA and LA suggesting that the decrease in production of PGE₂resulted from both the shift in the n-6:n-3 fatty acid ratio and fromDHA-mediated inhibition of AA hydrolysis.

[0086] Measurement ofeicosanoids: 2-series eicosanoids may be associatedwith neuronal apoptosis in age-associated neurodegenerative diseases andacute excitotoxic insults such as ischemia (Sanzgiri et al., J.Neurobiol. 41:221-229, 1999; Drachman and Rothstein, Ann. Neurol.48:792-795, 2000; Bezzi et al., Nature 391:281-285, 1998). Arachidonicacid (AA, 20:4n-6) and eicosapentaenoic acid (EPA, 20:5n-3) are theprecursors of 2- and 3-series of eicosanoids, respectively. To determinewhether the gene transfer-mediated alteration in the contents of AA andEPA may lead to a difference in the production of eicosanoids in thecells, we measured the production of prostaglandin E₂, one of the majoreicosanoids derived from AA, in infected cells after stimulation withcalcium ionophore A23187. More specifically, prostaglandin E₂ wasmeasured by using enzyme immunoassay kits (Cayman Chemical, Ann Arbor,Mich.) following the manufacturer's protocol. (The crossreactivity withprostaglandin E3 is 16%.) Cultured cells were washed with LH buffer(with 1% BSA) and incubated with the same buffer containing the calciumionophore A23187 (5 μM). After a 10-minute incubation, the conditionedbuffer was recovered and subjected to eicosanoid measurement. The amountof prostaglandin E₂ produced by fat-1 expressing cells was 20% lowerthan that produced by control cells (FIG. 11).

[0087] Induction of apoptosis and determination of cell growth andviability: Apoptosis was induced by growth factor withdrawal.Forty-eight hours after neurons were transfected, the culture media waschanged to Neurobasal™ Medium supplemented with 25 mM glutamic acid(Sigma Chemical Co., St. Louis, Mo.) and 0.5 mM glutainine. Cytotoxicitywas measured 24 hours after growth factors were withdrawn using theVybrant™ Apoptosis Assay (Molecular Probes, Eugene, Oreg.). Briefly,cells were washed with ice-cold phosphate buffered saline (PBS) andsubsequently incubated on ice for 20-30 min in ice-cold PBS containingHoechst 33342 solution (1 ml/ml) and PI solution (1 ml/ml). A photographwas taken at the end of the incubation period.

[0088] Cell growth and viability: Cell growth and viability weredetermined using the MTT cell proliferation kit (Roche DiagnosticCorporation). MTT labeling reagent (100 μl) was added to each well.After 4 hours of incubation, 1.0 ml of the solubilization solution wasadded into each well. The cells were then incubated overnight at 37° C.and the spectrophotometiical absorbency of the solution at 600 nm wasmeasured.

[0089] Expression of the fat-1 gene provided strong protection againstapoptosis in rat cortical neurons. Hoest 33625 and PI staining ofcortical cultures 24 hours after the induction of apoptosis, show thatcultures infected with Ad-GFP-fat-1 underwent less apoptosis than thoseinfected with Ad-GFP. MTT analysis indicated that the viability ofAd-GFP-fat-1 cells was significantly (p<0.05) higher than that of cellsinfected with Ad-GFP (FIG. 12). These results indicate that theexpression of fat-1 can inhibit neuronal apoptosis and promote cellviability. The ability of the C. elegans n-3 fatty acid desaturase toinhibit apoptosis of neuronal cells highlights the importance of then-6:n-3 fatty acid ratio in neuroprotection. Accordingly, techniquesthat deliver a fat-1 sequence, or a biologically active variant thereof,to neurons provide the means to quicldy and dramatically balancecellular n-6:n-3 fatty acid ratio, alter eicosanoid profile (and therebyexert an anti-apoptotic effect on neuronal cells) without the need forsupplementation with exogenous n-3 PUFAs. Compared to dietaryintervention, this approach is more effective in balancing the n-6:n-3ratio because it simultaneously elevates the tissue concentration of n-3PUFAs and reduces the level of endogenous n-6 PUFAs. This method is anovel and effective approach to modifying fatty acid composition inneuronal cells, and it can be applied as a stand-alone gene therapy oras an adjuvant therapy or chemopreventive procedure (in, for example,apoplexy patients).

[0090] Data analysis, statistical analysis: Cell viability data (MTT),as well as fatty acid composition and eicosanoids levels were comparedusing the Student t-test. The analysis included 6 wells/group (exceptlipid analyses; 4 wells/group) and each experiment was repeated 3 times.The level of significance was set at p<0.05.

Example 10 Fat-1 Expression in Human Endothelium and Inhibition ofInflammation

[0091] To determine whether the conversion of n-6 to n-3 PUFA can begenetically conferred to primary human vascular endothelium and to studyits potential protective effects against endothelial activation aftercytokine stimulation, a first generation (type 5) recombinant adenoviralvector (Ad) was constructed which contained the fat-1 transgene inseries with a GFP expression cassette under the control of the CMVpromoter (Ad.fat-1). A GFP/β-gal adenovirus served as the control vector(Ad.GFP/β-gal). Monolayers of primary human umbilical vein endothelialcells (HUVECs) were infected with Ad.fat-1 or the control Ad for 36hours, exposed for 24 hours to 10 mM arachidonic acid, and subjected tolipid analysis by gas chromatography, surface adhesion molecule analysisby inimunoassay, and videomicroscopy to study endothelial interactionswith the monocytic cell line, THP-1, under laminar flow conditions.

[0092] Expression of fat-1 dramatically altered the lipid composition ofhuman endothelial cells and changed the overall ratio of n-6 to n-3 PUFAfrom 8.5 to 1.4. Furthermore, after cytoline exposure (TNF-α, 5 μlapplied for 4 hours) fat-1 expression significantly reduced the surfaceexpression of the adhesion molecules and markers of inflammation(E-Selectin, ICAM-1, and VCAM-1 by 42%, 43%, and 57%, respectively(p<0.001)).

[0093] We then asked whether changes in the adhesion molecule profilewere sufficient to alter endothelial interactions with monocytes, themost prevalent white blood cell type found in atherosclerotic lesions.Under laminar flow and a defined shear stress of ˜2 dynes/cm²,fat-1-infected HUVEC, compared to control vector-infected HUVEC,supported ˜50% less finn adhesion with almost no effect on the rollinginteractions of THP-1 cells. Thus, heterologous expression of the C.elegans desaturase, fat-1, confers on human endothelial cells theability to convert n-6 to n-3 PUFA. This effect significantly repressedcytokine induction of the endothelial inflammatory response and firmadhesion of the monocytic cell line, THP-1, under simulatedphysiological flow conditions. Accordingly, expression of fat-1represents a potential therapeutic approach to treating inflanmiatoryvascular diseases, such as atherosclerosis.

Example 11 n-3 Desaturase as an Anti-Arrhythmic Agent

[0094] To deteriine whether fat-1 expression could provide ananti-arrhythmic effect, myocytes expressing the n-3 desaturase wereexamined for their susceptibility to arrhythmias induced byarrhythmogenic agents. Neonatal rat cardiac myocytes, grown on glasscoverslips and able to spontaneously beat, were infected withAd.GFP.fat-1 or Ad.GFP. Two days after infection, cells were transferredto a perfusion system and perfused with serum free medium containinghigh concentrations (5-10 mM) of calcium. These media arearrhythmogenic. During the perfusion process, myocyte contraction wasmonitored using a phase contrast microscope and video-monitoredge-detector. Following the high [Ca²⁺] (7.5 mM) challenge, the controlcells infected with Ad.GFP promptly exhibited an increased beating ratefollowed by spasmodic contractions or fibrillation whereas the cellsinfected with Ad.GFP.fat-1 could sustain regular beating. Thus, myocytesexpressing the n-3 desaturase show little, if any, susceptibility toarrhythmogenic stimuli (FIG. 13).

Example 12 Fat-1 Expression and Inhibition of Tumor Growth

[0095] To test the effect of the gene transfer on tumor growth in vivo,we have carried out a pilot experiment in two nude mice bearing humanbreast cancer xenografts (MDA-MB-231). One mouse was injectedintratumorally with 50 ml of Ad.GFP.fat-1 (1012 particles/ml) twiceevery other day. The other was injected with the control vector(Ad.GFP). The growth rate of the tumors was monitored for four weeks.The growth rate of the tumor treated with Ad.GFP.fat-1 appeared to beslower than that of the control tumor (FIG. 14).

Example 13 The Effect of Fat-1 Expression on Fatty Acid Composition andGrowth of Human Breast Cancer Cells in Culture

[0096] Construction of Recombinant Adenovirus (Ad): A recombinant Adcarrying the fat-1 cDNA was constructed as described previously (Kang etal., Proc. Natl. Acad. Sci. USA 98:4050-4054, 2001). Briefly, the fat-1cDNA in pCE8 (as described above) was excised from the plasmid with anEcoRI/KpnI double digest, inserted into a shutter vector and thensubjected to homologous recombination with an adenoviral backboneaccording to the methods of He et al. (Proc. Natl. Acad. Sci. USA95:2509-2514, 1998). Two first-generation type 5 recombinantadenoviruses were generated: Ad.GFP, which carries GFP as a reportergene under control of the CMV promoter, and Ad.GFP.fat-1, which carriesboth the fat-1 and GFP genes, each under the control of separate CMVpromoters. The recombinant viruses were prepared as high titer stocksthrough propagation in 293 cells, as described previously (Kang et al.,Proc. Natl. Acad. Sci. USA 98:4050-4054, 2001). The integrity of theconstructs was confirmed by enzymatic digestion and by DNA sequenceanalysis.

[0097] Cell Cultures and Infection with Ad.: MCF-7 cells were routinelymaintained in 1:1 (v/v) mixture of DMEM and Ham's F12 medium (JRH,Bioscience) supplemented with 5% fetal bovine serum (FBS) plusantibiotic solution (penicillin, 50 U/ml; streptomycin, 50 μg/ml) at 37°C. in a tissue culture incubator with 5% CO₂ and 98% relative humidity.Cells were infected with Ad for experiments when they were grown toabout 70% confluence by adding virus particles to medium without serum(3-5×10⁸ particles/ml). Initially, optimal viral concentration wasdetermined by using Ad.GFP to achieve an optimal balance of high geneexpression and low viral titer to minimize cytotoxicity. After a 24-hourincubation, the infection medium was replaced with normal culture mediumsupplemented with 10 μM 18:2n6 and 20:4n6. Forty-eight hours afterinfection, cells were used for analyses of gene expression, fatty acidcomposition, eicosanoid production, and cell proliferation andapoptosis.

[0098] RNA Analysis: The fat-1 transcripts were examined by ribonucleaseprotection assay using a RPA III™ kit (Ambion, Austin, Tex.). Briefly,total RNA was extracted from cultured cells using a RNA isolation kit(Qiagen) according to the manufacturer's protocol. The plasmidcontaining fat-1, pCE8, was linearized and used as transcriptiontemplate. Antisense RNA probes were transcribed in vitro using ³³P-UTPand T7 polymerase (Riboprobe™ System T7 kit, Promega), hybridized withthe total RNA extracted from the cancer cells, and digested with RNaseto remove non-hybridized RNA and probe. The protected RNA:RNA wasresolved in denaturing sequence gel and subjected to autoradiography. Aprobe targeting the GAPDH gene was used as an internal control.

[0099] The cells that were infected and expressed the transgene could bereadily identified by fluorescence microscopy since they co-expressedthe GFP (which exhibites bright fluorescence). Three days afterinfection, it was observed that about 60-70 percent of the cells wereinfected and expressed the transgene. Analysis of mRNA using aribonuclease protection assay showed that fat-1 mRNA was highly abundantin cells infected with Ad.GFP.fat-1, but was not detected in cellsinfected with Ad.GFP (control). This result indicates that theAd-mediated gene transfer could confer a high expression of fat-1 genein MCF-7 cells, which normally lack the gene.

[0100] Lipid Analysis: To examine the efficacy of the gene transfer inmodifying the fatty acid composition of the human MCF-7 cells, totalcellular lipids were extracted and analyzed by gas chromatograph afterinfection with the Ads and incubation with n-6 fatty acids for 2-3 days.The fatty acid composition of total cellular lipids was analyzed asdescribed (Kang et al., supra). Lipid was extracted withchloroform/methanol (2:1, vol/vol) containing 0.005% butylatedhydroxytoluene (BHT, as antioxidant). Fatty acid methyl esters wereprepared by using a 14% (wt/vol) BF3/methanol reagent. Fatty acid methylesters were quantified with GC/MS by using an HP-5890 Series II gaschromatograph equipped with a Supelcowax SP-10 capillary column(Supelco, Bellefonte, Pa.) attached to an HP-5971 mass spectrometer. Theinjector and detector are maintained at 260° C. and 280° C.,respectively. The oven program is maintained initially at 150° C. for 2min, then ramped to 200° C. at 10° C./min and held for 4 min, rampedagain at 5° C./min to 240° C., held for 3 min, and finally ramped to270° C. at 10° C./min and maintained for 5 min. Carrier gas-flow rate ismaintained at a constant 0.8 ml/min throughout. Total ion monitoring isperformed, encompassing mass ranges from 50-550 atomic mass units. Fattyacid mass is determined by comparing areas of various analyzed fattyacids to that of a fixed concentration of internal standard.

[0101] The expression of fat-1 cDNA in MCF-7 cells resulted inconversions of n-6 fatty acids to n-3 fatty acids, and a significantchange in the ratio of n-6/n-3 fatty acids. The fatty acid profiles areremarkably different between the control cells infected just with theAd.GFP and the cells infected with the Ad.GFP.fat-1 (FIG. 15). Cellsinfected with Ad.GFP had no change in their fatty acid profiles whencompared with noninfected cells. In the cells expressing the fat-1 cDNA(n-3 fatty acid desaturase), various n-6 fatty acids were convertedlargely to the corresponding n3 fatty acids, for example, 18:2n6 to18:3n3, 20:4n6 to 20:5n3, and 22:4n6 to 22:5n3. As a result, the fattyacid composition of the cells expressing fat-1 gene was changedsignificantly when compared with that of the control cells infected withAd.GFP (FIG. 15), with a large reduction of the n-6/n-3 ratio from 12 inthe control cells to 0.8 in the cells expressing the n-3 fatty aciddesaturase.

[0102] Measurement of Eicosanoids: It has been shown previously thatprostaglandin E2 (PGE2), one of the major ecosanoids derived from 20:4n6(arachidonic acid), is associated with cancer development (Rose andconnolly, Pharmacol. ther. 83:217-244, 1999; cave, Breast Cancer Res.Treat. 46:239-246, 1997). To determine whether the gene transfer-inducedalteration in the contents of arachidonic and eicosapentaenoic acids canchange the production of eicosanoids in the cells, we measured theproduction of PGE2 in the infected cells after stimulation with calciumionophore A23187 by using an enzyme immunoassay kit that specificallydetects prostaglandin E2 derived from AA with a 16% crossreactivity withprostaglandin E3 from EPA. More specifically, prostaglandin E₂ wasmeasured by using enzyme immunoassay kits (Assay Designs, Inc) followingthe manufacturer's protocol. (The cross-reactivity with PGE₃ is 16%).Cultured cells were washed with PBS containing 1% BSA and incubated withserum-free medium containing calcium ionophore A23187 (5 μM). After a10-minute incubation, the conditioned medium was recovered and subjectedto eicosanoid measurement. The amount of prostaglandin E₂ produced bythe fat-1 cells was significantly lower than that produced by thecontrol cells (FIG. 16).

[0103] Analysis of Cell Proliferation and Apoptosis: To determine theeffect of expression of the fat-1 gene on MCF-7 cell growth, cellproliferation and apoptosis following gene transfer were assessed.Routinely, cell morphology was examined by microscopy (dead cells appearto be detached, round and small) and total number of cell in each wellwas determined by counting the viable cells using a hemocytometer. Inaddition, cell proliferation was assessed using a MTT Proliferation KitI (Roche Diagnostics Corporation). Apoptotic cells were determined bynuclear staining with Vybrant™ Apoptosis Kit #5 (Molecular Probes)following the manufacturer's protocol.

[0104] A large number of the cells expressing fat-I gene underwentapoptosis, as indicated by morphological changes (small size with roundshape or fragmentation) and nuclear staining (bright blue). Statisticanalysis of apoptotic cell counts showed that 30-50% of cells infectedwith Ad.GFP.fat-1 were apoptotic whereas only 10% dead cells found inthe control cells (infected with Ad.GFP). MTT analysis indicated thatproliferative activity of cells infected with Ad.GFP.fat-1 wassignificantly lower than that of cells infected with Ad.GFP.Accordingly, the total number of viable cells in the cells infected withAd.GFP.fat-1 was about 30% less than that in the control cells. Theseresults are consistent with the proposition that fat-1 expression canserve as an anti-cancer agent.

[0105] Data analyses, statistical analyses: Data were presented as mean±SE. Student's T test was used to evaluate the difference between twovalues. The level of significance was set at p<0.05.Results

[0106] A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.

1 4 1 28 DNA Artificial Sequence Synthetically generated primer 1agaattcggc acgagccaag tttgaggt 28 2 28 DNA Artificial SequenceSynthetically generated primer 2 gcctgaggct ttatgcattc aacgcact 28 31391 DNA Caenorhabditis elegans CDS (13)...(1221) 3 caagtttgag gt atggtc gct cat tcc tca gaa ggg tta tcc gcc acg gct 51 Met Val Ala His SerSer Glu Gly Leu Ser Ala Thr Ala 1 5 10 ccg gtc acc ggc gga gat gtt ctggtt gat gct cgt gca tct ctt gaa 99 Pro Val Thr Gly Gly Asp Val Leu ValAsp Ala Arg Ala Ser Leu Glu 15 20 25 gaa aag gag gct cca cgt gat gtg aatgca aac act aaa cag gcc acc 147 Glu Lys Glu Ala Pro Arg Asp Val Asn AlaAsn Thr Lys Gln Ala Thr 30 35 40 45 act gaa gag cca cgc atc caa tta ccaact gtg gat gct ttc cgt cgt 195 Thr Glu Glu Pro Arg Ile Gln Leu Pro ThrVal Asp Ala Phe Arg Arg 50 55 60 gca att cca gca cac tgt ttc gaa aga gatctc gtt aaa tca atc aga 243 Ala Ile Pro Ala His Cys Phe Glu Arg Asp LeuVal Lys Ser Ile Arg 65 70 75 tat ttg gtg caa gac ttt gcg gca ctc aca attctc tac ttt gct ctt 291 Tyr Leu Val Gln Asp Phe Ala Ala Leu Thr Ile LeuTyr Phe Ala Leu 80 85 90 cca gct ttt gag tac ttt gga ttg ttt ggt tac ttggtt tgg aac att 339 Pro Ala Phe Glu Tyr Phe Gly Leu Phe Gly Tyr Leu ValTrp Asn Ile 95 100 105 ttt atg gga gtt ttt gga ttc gcg ttg ttc gtc gttgga cac gat tgt 387 Phe Met Gly Val Phe Gly Phe Ala Leu Phe Val Val GlyHis Asp Cys 110 115 120 125 ctt cat gga tca ttc tct gat aat cag aat ctcaat gat ttc att gga 435 Leu His Gly Ser Phe Ser Asp Asn Gln Asn Leu AsnAsp Phe Ile Gly 130 135 140 cat atc gcc ttc tca cca ctc ttc tct cca tacttc cca tgg cag aaa 483 His Ile Ala Phe Ser Pro Leu Phe Ser Pro Tyr PhePro Trp Gln Lys 145 150 155 agt cac aag ctt cac cat gct ttc acc aac cacatt gac aaa gat cat 531 Ser His Lys Leu His His Ala Phe Thr Asn His IleAsp Lys Asp His 160 165 170 gga cac gtg tgg att cag gat aag gat tgg gaagca atg cca tca tgg 579 Gly His Val Trp Ile Gln Asp Lys Asp Trp Glu AlaMet Pro Ser Trp 175 180 185 aaa aga tgg ttc aat cca att cca ttc tct ggatgg ctt aaa tgg ttc 627 Lys Arg Trp Phe Asn Pro Ile Pro Phe Ser Gly TrpLeu Lys Trp Phe 190 195 200 205 cca gtg tac act tta ttc ggt ttc tgt gatgga tct cac ttc tgg cca 675 Pro Val Tyr Thr Leu Phe Gly Phe Cys Asp GlySer His Phe Trp Pro 210 215 220 tac tct tca ctt ttt gtt cgt aac tct gaccgt gtt caa tgt gta atc 723 Tyr Ser Ser Leu Phe Val Arg Asn Ser Asp ArgVal Gln Cys Val Ile 225 230 235 tct gga atc tgt tgc tgt gtg tgt gca tatatt gct cta aca att gct 771 Ser Gly Ile Cys Cys Cys Val Cys Ala Tyr IleAla Leu Thr Ile Ala 240 245 250 gga tca tat tcc aat tgg ttc tgg tac tattgg gtt cca ctt tct ttc 819 Gly Ser Tyr Ser Asn Trp Phe Trp Tyr Tyr TrpVal Pro Leu Ser Phe 255 260 265 ttc gga ttg atg ctc gtc att gtt acc tatttg caa cat gtc gat gat 867 Phe Gly Leu Met Leu Val Ile Val Thr Tyr LeuGln His Val Asp Asp 270 275 280 285 gtc gct gag gtg tac gag gct gat gaatgg agc ttc gtc cgt gga caa 915 Val Ala Glu Val Tyr Glu Ala Asp Glu TrpSer Phe Val Arg Gly Gln 290 295 300 acc caa acc atc gat cgt tac tat ggactc gga ttg gac aca acg atg 963 Thr Gln Thr Ile Asp Arg Tyr Tyr Gly LeuGly Leu Asp Thr Thr Met 305 310 315 cac cat atc aca gac gga cac gtt gcccat cac ttc ttc aac aaa atc 1011 His His Ile Thr Asp Gly His Val Ala HisHis Phe Phe Asn Lys Ile 320 325 330 cca cat tac cat ctc atc gaa gca accgaa ggt gtc aaa aag gtc ttg 1059 Pro His Tyr His Leu Ile Glu Ala Thr GluGly Val Lys Lys Val Leu 335 340 345 gag ccg ttg tcc gac acc caa tac gggtac aaa tct caa gtg aac tac 1107 Glu Pro Leu Ser Asp Thr Gln Tyr Gly TyrLys Ser Gln Val Asn Tyr 350 355 360 365 gat ttc ttt gcc cgt ttc ctg tggttc aac tac aag ctc gac tat ctc 1155 Asp Phe Phe Ala Arg Phe Leu Trp PheAsn Tyr Lys Leu Asp Tyr Leu 370 375 380 gtt cac aag acc gcc gga atc atgcaa ttc cga aca act ctc gag gag 1203 Val His Lys Thr Ala Gly Ile Met GlnPhe Arg Thr Thr Leu Glu Glu 385 390 395 aag gca aag gcc aag taaaagaatatcc cgtgccgttc tagagtacaa 1251 Lys Ala Lys Ala Lys * 400caacaacttc tgcgttttca ccggttttgc tctaattgca atttttcttt gttctatata 1311tatttttttg ctttttaatt ttattctctc taaaaaactt ctacttttca gtgcgttgaa 1371tgcataaagc cataactctt 1391 4 402 PRT Caenorhabditis elegans 4 Met ValAla His Ser Ser Glu Gly Leu Ser Ala Thr Ala Pro Val Thr 1 5 10 15 GlyGly Asp Val Leu Val Asp Ala Arg Ala Ser Leu Glu Glu Lys Glu 20 25 30 AlaPro Arg Asp Val Asn Ala Asn Thr Lys Gln Ala Thr Thr Glu Glu 35 40 45 ProArg Ile Gln Leu Pro Thr Val Asp Ala Phe Arg Arg Ala Ile Pro 50 55 60 AlaHis Cys Phe Glu Arg Asp Leu Val Lys Ser Ile Arg Tyr Leu Val 65 70 75 80Gln Asp Phe Ala Ala Leu Thr Ile Leu Tyr Phe Ala Leu Pro Ala Phe 85 90 95Glu Tyr Phe Gly Leu Phe Gly Tyr Leu Val Trp Asn Ile Phe Met Gly 100 105110 Val Phe Gly Phe Ala Leu Phe Val Val Gly His Asp Cys Leu His Gly 115120 125 Ser Phe Ser Asp Asn Gln Asn Leu Asn Asp Phe Ile Gly His Ile Ala130 135 140 Phe Ser Pro Leu Phe Ser Pro Tyr Phe Pro Trp Gln Lys Ser HisLys 145 150 155 160 Leu His His Ala Phe Thr Asn His Ile Asp Lys Asp HisGly His Val 165 170 175 Trp Ile Gln Asp Lys Asp Trp Glu Ala Met Pro SerTrp Lys Arg Trp 180 185 190 Phe Asn Pro Ile Pro Phe Ser Gly Trp Leu LysTrp Phe Pro Val Tyr 195 200 205 Thr Leu Phe Gly Phe Cys Asp Gly Ser HisPhe Trp Pro Tyr Ser Ser 210 215 220 Leu Phe Val Arg Asn Ser Asp Arg ValGln Cys Val Ile Ser Gly Ile 225 230 235 240 Cys Cys Cys Val Cys Ala TyrIle Ala Leu Thr Ile Ala Gly Ser Tyr 245 250 255 Ser Asn Trp Phe Trp TyrTyr Trp Val Pro Leu Ser Phe Phe Gly Leu 260 265 270 Met Leu Val Ile ValThr Tyr Leu Gln His Val Asp Asp Val Ala Glu 275 280 285 Val Tyr Glu AlaAsp Glu Trp Ser Phe Val Arg Gly Gln Thr Gln Thr 290 295 300 Ile Asp ArgTyr Tyr Gly Leu Gly Leu Asp Thr Thr Met His His Ile 305 310 315 320 ThrAsp Gly His Val Ala His His Phe Phe Asn Lys Ile Pro His Tyr 325 330 335His Leu Ile Glu Ala Thr Glu Gly Val Lys Lys Val Leu Glu Pro Leu 340 345350 Ser Asp Thr Gln Tyr Gly Tyr Lys Ser Gln Val Asn Tyr Asp Phe Phe 355360 365 Ala Arg Phe Leu Trp Phe Asn Tyr Lys Leu Asp Tyr Leu Val His Lys370 375 380 Thr Ala Gly Ile Met Gln Phe Arg Thr Thr Leu Glu Glu Lys AlaLys 385 390 395 400 Ala Lys

What is claimed is:
 1. An isolated nucleic acid molecule comprising afirst and a second nucleic acid sequence, wherein the first sequenceencodes an enzyme that desaturates an ω-6 fatty acid to a correspondingω-3 fatty acid and the second sequence is a tissue-specific promoterthat directs expression of the first sequence in a selected mammaliancell type.
 2. The nucleic acid molecule of claim 1, wherein themammalian cell type is a myocyte, endothelial cell, adipose cell, orneuron.
 3. The nucleic acid molecule of claim 1, wherein the mammaliancell type is a type of cancer cell.
 4. An expression vector comprisingthe nucleic acid molecule of claim
 1. 5. A mammalian cell comprising thevector of claim
 4. 6. A transgenic mammal comprising the cDNA sequenceshown in FIG. 17A, or a biologically active fragment or mutant thereof.7. A method of improving the content of ω-3 fatty acids in a patient'sdiet, the method comprising providing to the patient a food productobtained from a transgenic manmmal that expresses a fat-1 gene of anon-mammalian animal, or a biologically active fragment or variantthereof.
 8. A method of treating a patient who has cancer, the methodcomprising administering to the patient a therapeutically effectiveamount of the nucleic acid molecule of claim 1, wherein thetissue-specific promoter directs expression of the sequence encoding theenzyme in the patient's cancerous cells.
 9. The method of claim 8,wherein the cancerous cells are breast cancer cells or colon cancercells.
 10. A method of inhibiting neuronal cell death in a patient, themethod comprising administering to the patient a therapeuticallyeffective amount of the nucleic acid molecule of claim 1, wherein thetissue-specific promoter directs expression of the sequence encoding theenzyme in neurons.
 11. The method of claim 10, wherein the patient has aneurodegenerative disease.
 12. The method of claim 11, wherein theneurodegenerative disease is Alzheimer's disease, Parkinson's disease,or Huntington's disease.
 13. A method of treating a patient who has, orwho may develop, a condition associated with an insufficiency of n-3polyunsaturated fatty acid (PUFA) or an imbalance in the ratio ofn-3:n-6 PUFAs, the method comprising administering to the patient anucleic acid encoding an n-3 desaturase or a biologically active variantthereof.
 14. The method of claim 13, wherein the condition is anarrhythmia, cardiovascular disease, cancer, an inflammatory disease, anautoimmune diseases, a malformation or threatened malformation of theretina or brain, diabetes, obesity, a sldn disorder, a renal disease,ulcerative colitis, Crohn's disease, or chronic obstructive pulmonarydisease.
 15. A method of treating a patient who has received, or who isscheduled to receive, a transplant comprising a biological organ,tissue, or cell, the method comprising administering to either thepatient or to the transplant, a nucleic acid encoding an n-3 desaturaseor a biologically active variant thereof.